1. Field of the Invention
The present invention relates to a multi-purpose promoter that can be employed to direct the expression of desired genes in genetically engineered plant cells or plants. The invention also provides the expression cassettes and plasmids comprising the 5xe2x80x2 untranscribed region of a TCTP gene from Arabidopsis. The reporter gene placed under the control of said promoter in the expression cassettes is ubiquitously expressed to an extremely high level in transgenic plants. In addition, the activity of said promoter is further induced by drought stress. The present invention also provides methods and processes for the preparation of the expression cassettes and transgenic plants comprising the expression cassettes.
2. Description of the Prior Art
In eukaryotic systems gene expression is regulated through complex interactions between various cis-acting and trans-acting regulatory elements. The cis-acting elements are defined as DNA sequences adjacent to a gene that directly or indirectly regulate the expression of the gene by modulating the binding of a variety of trans-acting elements, so called transcription factors. The promoter is one of the most important cis-acting elements and generally considered as a nucleotide sequence region that is located 5xe2x80x2 upstream to the transcription start site of a gene. The promoter contains the binding site for RNA polymerase II and initiates the transcription of the gene. It also contains several well-conserved sequence elements, such as the TATA box around base position xe2x88x9230 and the CAAT box around base position xe2x88x9270 relative to the transcription start site (+1) (Joshi 1987). The TATA box is essential for accurately positioning the initiation of transcription. The CAAT box is not present in all promoters but is in some promoters replaced with a GC-rich element. In addition to these basic elements, the promoter also contains additional sequence elements that are responsive to environmental signals and to physiological conditions, such as drought stress, heat shock, cold stress, availability of nutrients, light, and ion strength. Some promoters also contain sequence elements that confer tissue- or cell-specific gene expression. The promoter is also critical in plant genetic engineering in that a gene of interest can be expressed in an important agronomic plant in ways that the gene expression is modulated in terms of expression level, tissue- or cell-specificities, developmental stage dependence, and responsiveness to a specific environmental stimulus.
Translationally controlled tumor protein (TCTP) is one of the recently identified growth-related proteins in plants and animals. It is a highly conserved cytosolic protein among various organisms, including man, animals, plants, and yeast (Woo et al. 1997) and very stable against heat, pH, ionic strength, and even against protease, suggesting that it has a very compact globular structure. One interesting characteristic of the TCTP protein is that its expression is closely related with growth condition of cells. In accordance with this, the TCTP protein has been originally isolated from rapidly growing tissues, including cancerous tissue in animals and callus tissue, apical stem and leaf, and root meristem tissue in plants. These observations suggest that it may have a regulatory role in cell proliferation (Woo et al. 1997; MacDonald et al. 1995; Hughes et al. 1993). However it has been later found that the TCTP protein is also expressed in healthy animal and plant tissues and that the expression is regulated by calcium ion at both the transcriptional and post-transcriptional levels (Wu et al. 1999; Sanchez et al. 1997; Xu et al. 1999). The TCTP protein associates with cytoskeletal microtubular networks (Gachet et al. 1999; Gachet et al. 1997) via direct interaction with xcex1- and xcex2-tubulins in a Ca2+-dependent manner in animal cells. As a whole the TCTP protein is an acidic protein with a pI of about 4.0, but a domain of about 50 amino acids in the central region is highly basic with a pI of 9.4. This basic domain physically interacts with the tubulins (Gachet et al. 1999). Taken together, these observations propose that the TCTP protein has a housekeeping role in the regulation of cell growth and differentiation. Many TCTP genes and gene sequences have been isolated from various tissues in animals and plants (Sage-Ono et al. 1998; Tamaoki et al. 1997; Woo et al. 1997). However, except for a few cases, only the TCTP gene sequences have been deposited in the databases without detailed molecular biological and functional analysis, especially in plants. A pea TCTP gene is expressed to a high level in rapidly dividing cells within the root cap (Woo et al. 1997). In a short-day plant Japanese morning glory (Pharbitis nil cv. Violet), the TCTP mRNA accumulates to a high level when the plant is grown in the dark, but the expression level decreases to an undetectable level in the light (Sage-Ono et al. 1998), suggesting that the TCTP has a role in the light-regulated growth at least in this plant species. Except for these two cases, no detailed studies have been reported in plants.
We recently found that a pea TCTP protein directly interacts with a Rab-like small molecular weight GTPase (small GTPase), pea Pra3 that is light-repressible and predominantly expressed in the epicotyls of dark-grown pea seedlings. Northern blot analysis revealed that the pea TCTP gene is transcribed to a high level in all plant parts examined, such as stems, leaves, and roots, but with the highest transcript level in the meristem tissue of root tip. In addition, the transcription was further induced with a factor of 2-3 by a 15% PEG treatment that mimics the drought stressed condition in plants. The same expression patterns were also observed with a tobacco TCTP gene homolog. These results, taken together with the previous reports (Sage-Ono et al. 1998; Woo et al. 1997), suggested that the promoter of the plant TCTP genes would be useful for the expression of foreign genes of interest in transgenic plants with high economic value. In an attempt to investigate the regulatory mechanism on the TCTP gene expression in plants and to evaluate the potential of the TCTP promoter in plant genetic engineering, we isolated a DNA sequence from the 5xe2x80x2 untranscribed region of a TCTP gene from Arabidopsis, defined the promoter region by serial deletions, and examined the expression level and pattern of the reporter gene placed under the control of the TCTP promoter in transgenic plants.
The Arabidopsis TCTP promoter region contains a typical TATA consensus sequence at base position xe2x88x92120 relative to the ATG start codon. However, neither CAAT box nor GC-rich element is identified at appropriate positions in the 5xe2x80x2 untranscribed region. Consistent with the previous observations, a couple of light-responsive elements are identified. The TCTP promoter led to a high-level expression of a reporter gene in all plant parts with the highest transcription level in the meristem tissue of root tip, which is consistent with the previous result in pea plant (Woo et al. 1997). Several different TCTP promoter constructs with deletions from the 5xe2x80x2 end were linked to the reporter gene, and the expression cassettes were examined in transgenic Arabidopsis plants. A minimum of 300 bp was enough for the high-level expression of the reporter gene. In addition, the 300-bp promoter element exhibited the most predominantly localized expression in the root meristem. These characteristics indicate that the TCTP promoter can be utilized to drive a high-level expression of genes of interest in appropriate host plants.
With recent technical developments and advances in plant tissue culture and plant genetic engineering, it is now a routine experiment to introduce a useful gene into a desired host plant with an aim to improve productivity and quality that can""t be easily achieved by classical breeding. To achieve this goal, a foreign gene is precisely linked to the TCTP promoter to make the gene expressible under the control of such promoter in transgenic plants.
The present invention provides a nucleic acid molecule from the 5xe2x80x2 untranscribed region of an Arabidopsis TCTP gene that ensures the high-level expression of a foreign gene properly linked to said nucleic acid molecule in transgenic plants. With the present invention, it is possible to express a gene of interest to a high level in whole plant as well as selectively in the meristem tissue of root tip.
Any desired gene is placed under the control of the TCTP promoter in an appropriate expression cassette, and the recombinant expression cassette is introduced into a host plant by various transfection methods, well-known techniques in the art. The promoter in this invention is also responsive to and further induced with a factor of 2-3 by drought stress. Regarding this, the present invention can be particularly utilized to engineer a gene of interest to be expressed to a certain level under normal condition and to be further induced by drought stress.
Root growth is critical for the yield of agronomic plants. The TCTP promoter according to the present invention can be used to accelerate root growth, especially under drought stress or water deficit. In one aspect of the invention, the TCTP promoter can be utilized to express genes involved in the stimulation and regulation of root growth and development.
The present invention also provides the expression cassettes and plasmids comprising the TCTP promoter as described above that are introduced into genetically engineered plants.
Another aspect of the present invention relates to transgenic plant cells and plants containing any gene of interest placed under the control of the TCTP promoter and to experimental processes to isolate the TCTP promoter sequence from Arabidopsis plant.
With the present invention, it is possible to engineer a plant so that it can produce a large amount of a biologically important material, exhibit improved growth rate and development, or show enhanced adaptability to environmental changes.